Project Summary/Abstract Antiviral therapies for the lifelong treatment of HIV has been established to cause nephrotoxic or cardiotoxic effects. Because there is currently not a cure for HIV, this poses a problem because the toxicity may affect patient adherence to these drug regimens. It has been observed that nucleoside reverse transcriptase inhibitors (NRTIs) cause mitochondrial dysfunction, leading to toxicity within the kidneys or heart. A validated mechanism of toxicity is through the incorporation of NRTIs by DNA polymerase ?, the primary replicative polymerase within the mitochondria, compromising replication. However, this mechanism is validated only for a subset of NRTIs, suggesting that other polymerases may play a role in mediating mitochondrial toxicity. The proposed project explores PrimPol, a recently characterized polymerase found in the mitochondria implicated in replication restart and translesion synthesis, as a route for NRTI-associated toxicity. The project particularly addresses this issue in the context of tenofovir, a widely used NRTI in modern treatments such as Atripla or Truvada, in which the mechanism of nephrotoxicity has not yet been established. In the first aim, enzymatic incorporation assays and X-ray crystallography will be used to determine the biochemical and structural basis for tenofovir incorporation by PrimPol. In the second aim, PrimPol overexpression and shRNA knockdown strains will be generated from HEK cells and renal proximal tubular epithelial cells to test the association of PrimPol with mitochondrial toxicity that is observed in patients when treated with tenofovir. In conjunction, these aims will establish the mechanism of toxicity of antiviral therapies and improve the development of nucleoside analog inhibitors with better pharmacological profiles.